Web‐Based Analysis and Publication of Flow Cytometry Experiments
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- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
Cytobank is a Web?based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. Curr. Protoc. Cytom. 53:10.17.1?10.17.24. © 2010 by John Wiley & Sons, Inc.
Keywords: Cytobank; database; storage platform; analysis; flow cytometry
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Online Storage of Flow Cytometry Data
- Basic Protocol 2: Analysis of Phospho‐Flow Signaling Experiments
- Basic Protocol 3: Annotating and Gating Experiments
- Basic Protocol 4: Sharing Illustrations and Data with Collaborators
- Basic Protocol 5: Publishing Experimental Data and Figures
- Basic Protocol 6: Initiating New Analysis of a Published Dataset
- Commentary
- Literature Cited
- Figures
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Figure 10.17.1 Searching the Experiment Inbox for the Welcome to Cytobank—U937 dataset experiment. In the upper‐right‐hand corner search box, type “welcome” to filter the experiments and show only those that match that text (https://www.cytobank.org/cytobank/experiments). View Image -
Figure 10.17.2 A histogram overlay figure on Cytobank. This figure shows the response of the U937 cell line to various stimulation conditions (in rows) at different signaling node readouts (p‐STAT6, p‐STAT3, p‐STAT1, and p‐STAT5, in columns). Histogram overlays on Cytobank shade peaks according to a statistic defined by the researcher. In this histogram overlay of a signaling experiment, the peaks are shaded by the fold change of each stimulated sample from the unstimulated control (in the first row). Each column shows intracellular detection of a phosphorylated STAT protein (1). Also included is a table of the statistics and a color scale (https://www.cytobank.org/cytobank/experiments/61/illustrations/96#Illustration). View Image -
Figure 10.17.3 Downloading files from Cytobank. To download files from Cytobank, click the Download FCS Files link on the experiment summary page (step 6) and follow the instructions to select a folder on your personal computer and start the download. This download will retrieve entire files in FCS or text format. Download of gated subpopulations can be done from within illustrations (https://www.cytobank.org/cytobank/experiments/61/download_files). View Image -
Figure 10.17.4 Creating a new experiment on Cytobank. Prior to upload, organize your files into groups by experiment. Give each experiment a name and assign it to a project. Also describe a short purpose for the experiment. These details can be searched later, if you are trying to find an experiment (https://www.cytobank.org/cytobank/experiments/new). View Image -
Figure 10.17.5 Successful file upload. Following file upload, you should see this message and links to either start an illustration or view the experiment summary page. Below this message you will see your files categorized and a plot of forward and side scatter for all the files to make sure each file is readable. View Image -
Figure 10.17.6 This figure shows the configuration of the controls and the resulting contour plot illustration at the end of the Contour Plot steps. (A ) Configuration of the controls to make this illustration. All the dosages are selected and this dimension is shown in the columns. For other dimensions, only one entry is selected to keep the figure small. (B ) Contour plots show an IL‐2 dose response where p‐STAT5 was measured in CD3+ T cells. The x ‐axis shows CD4 expression and the y ‐axis shows phosphorylation of STAT5. The doses are arranged in columns, starting with unstimulated cells, then a dose response from 100 ng/ml to 0.01 ng/ml (https://www.cytobank.org/cytobank/experiments/310/illustrations/583#Illustration). View Image -
Figure 10.17.7 Setup of the figure dimensions for a histogram overlay comparing dosages. When the plot type is a histogram overlay, the second dimension is “Overlaid”, which means that it forms the overlaid peaks within the histogram overlay. Usually the overlaid dimension compares conditions, individuals, time‐points, or dosages. View Image -
Figure 10.17.8 A histogram overlay showing the dose response for CD3+ T cells from two healthy blood donors. Along with the histogram overlay is a table of data showing the fold change calculations (https://www.cytobank.org/cytobank/experiments/310/illustrations/584#Illustration). View Image -
Figure 10.17.9 A table of fold change statistics exported from Cytobank. This table of statistics compares the median fold change of each peak of CD3+ T cells stimulated with a different dose of IL‐2, relative to the unstimulated state. View Image -
Figure 10.17.10 Order of the dimension controls to create a heatmap of doses. For a heatmap, the dosages were moved into the columns and the individuals moved into the rows. View Image -
Figure 10.17.11 Placing the mouse over a square of a heatmap shows a view of the underlying data. Shown here is a heatmap view of the dose‐response experiment. Here, the mouse is moved over one square for 0.07 ng/ml IL‐2 in Donor 2, showing a 2‐D density dot plot view of the data used to generate the statistic in that square. This type of view‐through keeps users connected to the data, while also providing a high‐level summary of the results (https://www.cytobank.org/cytobank/experiments/310/illustrations/585#Illustration). View Image -
Figure 10.17.12 Web‐based gating. This figure shows the drawing of a polygon gate around intact cells using a plot of side scatter versus forward scatter (https://www.cytobank.org/cytobank/experiments/310/draw_gates). View Image -
Figure 10.17.13 An automatically generated layout checking whether the Intact Cells gate ‘fits’ for all of the files in the Phospho‐Flow Cytokine Titration experiment. In this case, one global Intact Cells gate is a good fit for all the files and identifies ∼90% of the events as intact cells in all the files (https://www.cytobank.org/cytobank/gates/check_gates?experimentID=310&gateID=1&populationID=0&plotType=14). View Image -
Figure 10.17.14 A hierarchy of the populations of cells present within the peripheral blood samples from the Phospho‐Flow Cytokine Titration experiment. This figure is a composite of two figures created on Cytobank—the Cell Populations hierarchy is viewable within gating, and the gating hierarchy plots are available within the illustrations by opening the Gating Hierarchy panel below the Figure Dimensions. View Image -
Figure 10.17.15 Clicking the Individuals button activates this dimension for annotation. The dimension is initially red, indicating that files need to be annotated for “which individual this file matches.” View Image -
Figure 10.17.16 Annotating information about Individuals. The tags Donor 1 and Donor 2 are entered as a comma‐delimited list into the Individuals annotation page. This creates two individual entries (Donor 1 and Donor 2) and looks for files with matching text in the filename or sample ID. View Image
Videos
Literature Cited
| Barrett, T., Troup, D.B., Wilhite, S.E., Ledoux, P., Rudnev, D., Evangelista, C., Kim, I.F., Soboleva, A., Tomashevsky, M., Marshall, K.A., Phillippy, K.H., Sherman, P.M., Muertter, R.N., and Edgar, R. 2009. NCBI GEO: Archive for high‐throughput functional genomic data. Nucleic Acids Res. 37:885‐890. | |
| Demeter, J., Beauheim, C., Gollub, J., Hernandez‐Boussard, T., Jin, H., Maier, D., Matese, J.C., Nitzberg, M., Wymore, F., Zachariah, Z.K., Brown, P.O., Sherlock, G., and Ball, C.A. 2007. The Stanford Microarray Database: Implementation of new analysis tools and open source release of software. Nucleic Acids Res. 35:766‐770. | |
| Frelinger, J., Kepler, T.B., and Chan, C. 2008. Flow: Statistics, visualization and informatics for flow cytometry. Biol. Med. 3:10. | |
| Hahne, F., LeMeur, N., Brinkman, R.R., Ellis, B., Haaland, P., Sarkar, D., Spidlen, J., Strain, E., and Gentleman, R. 2009. FlowCore: A bioconductor package for high throughput flow cytometry. BMC Bioinformatics 10:106. | |
| Irish, J.M., Hovland, R., Krutzik, P.O., Perez, O.D., Bruserud, Ø., Gjertsen, B.T., and Nolan, G.P. 2004. Single cell profiling of potentiated phospho‐protein networks in cancer cells. Cell 118:217‐228. | |
| Irish, J.M., Kotecha, N., and Nolan, G.P. 2006. Mapping normal and cancer cell signaling networks: Towards single‐cell proteomics. Nat. Rev. Cancer 6:146‐155. | |
| Krutzik, P.O. and Nolan, G.P. 2003. Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry A 55:61‐70. | |
| Krutzik, P.O. and Nolan, G.P. 2006. Fluorescent cell barcoding in flow cytometry allows high‐throughput drug screening and signaling profiling. Nat. Methods 3:361‐368. | |
| Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A., and Nolan, G.P. 2005. Causal protein‐signaling networks derived from multiparameter single‐cell data. Science 308:523‐529. | |
| Internet Resources | |
| http://www.cytobank.org | |
| This is the home page for Cytobank with links for logging in and registration, getting started, and to the latest documentation. | |
| http://docs.cytobank.org | |
| This is the documentation site for Cytobank with links to the latest tutorials and frequently asked questions. | |
| http://www.mozilla.org | |
| This is the homepage for Mozilla, creators of the FireFox Web browser. Cytobank works best with FireFox. | |
| http://www.java.com | |
| This is the homepage for Java, with links and information about installing Java on your computer. Cytobank requires that your Web browser be configured to run java applets. | |
| http://www.javatester.org/version.html | |
| This site can test whether your browser can run Java applets. |
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